QC Report

	general
Report generated at	2023-03-11 10:39:17
Title	ht22 ATAC-seq mini-encode
Description	ht22 ATAC-seq mini-encode test run
Pipeline version	v2.2.1
Pipeline type	atac
Genome	mm10
Aligner	bowtie2
Sequencing endedness	{'rep1': {'paired_end': True}, 'rep2': {'paired_end': True}, 'rep3': {'paired_end': True}}
Peak caller	macs2

Alignment quality metrics

SAMstat (raw unfiltered BAM)

	rep1	rep2	rep3
Total Reads	301779192	256710874	162286922
Total Reads (QC-failed)	0	0	0
Duplicate Reads	0	0	0
Duplicate Reads (QC-failed)	0	0	0
Mapped Reads	294205880	248809664	156910174
Mapped Reads (QC-failed)	0	0	0
% Mapped Reads	97.5	96.89999999999999	96.7
Paired Reads	301779192	256710874	162286922
Paired Reads (QC-failed)	0	0	0
Read1	150889596	128355437	81143461
Read1 (QC-failed)	0	0	0
Read2	150889596	128355437	81143461
Read2 (QC-failed)	0	0	0
Properly Paired Reads	292527390	243826914	154325150
Properly Paired Reads (QC-failed)	0	0	0
% Properly Paired Reads	96.89999999999999	95.0	95.1
With itself	292569910	245964602	155121700
With itself (QC-failed)	0	0	0
Singletons	1635970	2845062	1788474
Singletons (QC-failed)	0	0	0
% Singleton	0.5	1.0999999999999999	1.0999999999999999
Diff. Chroms	3091	161783	51041
Diff. Chroms (QC-failed)	0	0	0

Marking duplicates (filtered BAM)

	rep1	rep2	rep3
Unpaired Reads	0	0	0
Paired Reads	112389616	94248968	59862901
Unmapped Reads	0	0	0
Unpaired Duplicate Reads	0	0	0
Paired Duplicate Reads	38514725	14286811	8365304
Paired Optical Duplicate Reads	743516	928340	462407
% Duplicate Reads	34.2689	15.1586	13.9741

Filtered with samtools flag 1804 (samtools view -F 1804):

read unmapped (0x4)
mate unmapped (0x8, for paired-end)
not primary alignment (0x100)
read fails platform/vendor quality checks (0x200)
read is PCR or optical duplicate (0x400)

Fraction of mitochondrial reads (unfiltered BAM)

	rep1	rep2	rep3
Rn = Number of Non-mitochondrial Reads	292681750	247276636	156369615
Rm = Number of Mitochondrial Reads	4207083	4134932	1470032
Rm/(Rn+Rm) = Frac. of mitochondrial reads	0.01417056666459395	0.016446864529320305	0.009313452151853837

SAMstat (filtered/deduped BAM)

	rep1	rep2	rep3
Total Reads	147487386	159268434	102667862
Total Reads (QC-failed)	0	0	0
Duplicate Reads	0	0	0
Duplicate Reads (QC-failed)	0	0	0
Mapped Reads	147487386	159268434	102667862
Mapped Reads (QC-failed)	0	0	0
% Mapped Reads	100.0	100.0	100.0
Paired Reads	147487386	159268434	102667862
Paired Reads (QC-failed)	0	0	0
Read1	73743693	79634217	51333931
Read1 (QC-failed)	0	0	0
Read2	73743693	79634217	51333931
Read2 (QC-failed)	0	0	0
Properly Paired Reads	147487386	159268434	102667862
Properly Paired Reads (QC-failed)	0	0	0
% Properly Paired Reads	100.0	100.0	100.0
With itself	147487386	159268434	102667862
With itself (QC-failed)	0	0	0
Singletons	0	0	0
Singletons (QC-failed)	0	0	0
% Singleton	0.0	0.0	0.0
Diff. Chroms	0	0	0
Diff. Chroms (QC-failed)	0	0	0

Filtered and duplicates are removed. Subsampling with atac.subsample_reads is not done in alignment steps. Nodup BAM is converted into a BED type (TAGALIGN) later and then TAGALIGN is subsampled with such parameter in the peak-calling step.

Fragment length statistics (filtered/deduped BAM)

	rep1	rep2	rep3
Fraction of reads in NFR	0.9993395896975993	0.8476954749632342	0.9199384409576455
Fraction of reads in NFR (QC pass)	True	True	True
Fraction of reads in NFR (QC reason)	OK	OK	OK
NFR / mono-nuc reads	1882.6752072343631	5.803496499665853	11.768652859021797
NFR / mono-nuc reads (QC pass)	True	True	True
NFR / mono-nuc reads (QC reason)	OK	OK	OK
Presence of NFR peak	True	True	True
Presence of Mono-Nuc peak	False	False	False
Presence of Di-Nuc peak	True	False	False

Open chromatin assays show distinct fragment length enrichments, as the cut sites are only in open chromatin and not in nucleosomes. As such, peaks representing different n-nucleosomal (ex mono-nucleosomal, di-nucleosomal) fragment lengths will arise. Good libraries will show these peaks in a fragment length distribution and will show specific peak ratios.

NFR: Nucleosome free region

Sequence quality metrics (filtered/deduped BAM)

Open chromatin assays are known to have significant GC bias. Please take this into consideration as necessary.

Annotated genomic region enrichment

	rep1	rep2	rep3
Fraction of Reads in universal DHS regions	0.36001564906710054	0.36051699359334444	0.3796856995035116
Fraction of Reads in blacklist regions	0.0009586650345813302	0.0016784430742880288	0.00201200254856773
Fraction of Reads in promoter regions	0.02446430232345429	0.02795852817891083	0.03228157220221455
Fraction of Reads in enhancer regions	0.3335907858587988	0.3302254481889362	0.34447562568313733

Signal to noise can be assessed by considering whether reads are falling into known open regions (such as DHS regions) or not. A high fraction of reads should fall into the universal (across cell type) DHS set. A small fraction should fall into the blacklist regions. A high set (though not all) should fall into the promoter regions. A high set (though not all) should fall into the enhancer regions. The promoter regions should not take up all reads, as it is known that there is a bias for promoters in open chromatin assays.

Library complexity quality metrics

Library complexity (filtered non-mito BAM)

	rep1	rep2	rep3
Total Fragments	110773147	92609434	59293451
Distinct Fragments	75193012	80125720	51614861
Positions with Two Read	16055348	9296068	5782986
NRF = Distinct/Total	0.678802	0.8652	0.870499
PBC1 = OneRead/Distinct	0.684379	0.866793	0.871808
PBC2 = OneRead/TwoRead	3.205195	7.471163	7.781147

Mitochondrial reads are filtered out by default. The non-redundant fraction (NRF) is the fraction of non-redundant mapped reads in a dataset; it is the ratio between the number of positions in the genome that uniquely mapped reads map to and the total number of uniquely mappable reads. The NRF should be > 0.8. The PBC1 is the ratio of genomic locations with EXACTLY one read pair over the genomic locations with AT LEAST one read pair. PBC1 is the primary measure, and the PBC1 should be close to 1. Provisionally 0-0.5 is severe bottlenecking, 0.5-0.8 is moderate bottlenecking, 0.8-0.9 is mild bottlenecking, and 0.9-1.0 is no bottlenecking. The PBC2 is the ratio of genomic locations with EXACTLY one read pair over the genomic locations with EXACTLY two read pairs. The PBC2 should be significantly greater than 1.

Fragment: read for a single-ended dataset, pair of reads for a paired-ended dataset
NRF: non redundant fraction
PBC1: PCR Bottleneck coefficient 1
PBC2: PCR Bottleneck coefficient 2
PBC1 is the primary measure. Provisionally

0-0.5 is severe bottlenecking
0.5-0.8 is moderate bottlenecking
0.8-0.9 is mild bottlenecking
0.9-1.0 is no bottlenecking

Replication quality metrics

IDR (Irreproducible Discovery Rate) plots

Reproducibility QC and peak detection statistics

	overlap	idr
Nt	116462	18762
N1	136101	20120
N2	126570	19757
N3	104949	15352
Np	168705	36864
N optimal	168705	36864
N conservative	116462	18762
Optimal Set	pooled-pr1_vs_pooled-pr2	pooled-pr1_vs_pooled-pr2
Conservative Set	rep1_vs_rep3	rep2_vs_rep3
Rescue Ratio	1.4485840875135236	1.9648225135913016
Self Consistency Ratio	1.2968298888031329	1.310578426263679
Reproducibility Test	pass	pass

Reproducibility QC

N1: Replicate 1 self-consistent peaks (comparing two pseudoreplicates generated by subsampling Rep1 reads)
N2: Replicate 2 self-consistent peaks (comparing two pseudoreplicates generated by subsampling Rep2 reads)
Ni: Replicate i self-consistent peaks (comparing two pseudoreplicates generated by subsampling RepX reads)
Nt: True Replicate consistent peaks (comparing true replicates Rep1 vs Rep2)
Np: Pooled-pseudoreplicate consistent peaks (comparing two pseudoreplicates generated by subsampling pooled reads from Rep1 and Rep2)
Self-consistency Ratio: max(N1,N2) / min (N1,N2)
Rescue Ratio: max(Np,Nt) / min (Np,Nt)
Reproducibility Test: If Self-consistency Ratio >2 AND Rescue Ratio > 2, then 'Fail' else 'Pass'

Number of raw peaks

	rep1	rep2	rep3
Number of peaks	299456	299470	299458

The number of peaks is capped at 300000
Peaks are called from macs2 with p-val threshold 0.01

Peak calling statistics

Peak region size

	rep1	rep2	rep3	idr_opt	overlap_opt
Min size	150.0	150.0	150.0	160.0	150.0
25 percentile	178.0	185.0	171.0	316.0	231.0
50 percentile (median)	211.0	245.0	221.0	448.0	324.0
75 percentile	301.0	369.0	332.0	630.0	475.0
Max size	2340.0	2457.0	2086.0	2452.0	3214.0
Mean	261.484181315452	305.6386950278826	277.94504738561	509.5519748263889	387.6935360540589

Enrichment / Signal-to-noise ratio

Strand cross-correlation measures (filtered BAM)

	rep1	rep2	rep3
Number of Subsampled Reads	12500000	12500000	12500000
Estimated Fragment Length	0	0	0
Cross-correlation at Estimated Fragment Length	0.17309035333131	0.182730706614741	0.180868064800158
Phantom Peak	40	65	50
Cross-correlation at Phantom Peak	0.176399	0.1687966	0.1692393
Argmin of Cross-correlation	1500	1500	1500
Minimum of Cross-correlation	0.15066	0.1597217	0.1594195
NSC (Normalized Strand Cross-correlation coeff.)	1.148881	1.144057	1.134542
RSC (Relative Strand Cross-correlation coeff.)	0.8714526	2.535462	2.18422

Performed on subsampled (25000000) reads. Such FASTQ trimming is for cross-corrleation analysis only.

Fragment = read (for single-ended dataset) or pair of reads (for paired-ended dataset)
Normalized strand cross-correlation coefficient (NSC) = col9 in outFile
Relative strand cross-correlation coefficient (RSC) = col10 in outFile
Estimated fragment length = col3 in outFile, take the top value

TSS enrichment (filtered/deduped BAM)

	rep1	rep2	rep3
TSS enrichment	3.122141923303737	6.3331474449049425	4.79470448688959

Open chromatin assays should show enrichment in open chromatin sites, such as TSS's. An average TSS enrichment in human (hg19) is above 6. A strong TSS enrichment is above 10. For other references please see https://www.encodeproject.org/atac-seq/

Jensen-Shannon distance (filtered/deduped BAM)

	rep1	rep2	rep3
AUC	0.25765593542304766	0.2838827661987303	0.27447123443696314
Synthetic AUC	0.49628349055259524	0.4971632444397567	0.4962147028865357
X-intercept	0.15902905671167847	0.14314194816543388	0.15556768668102405
Synthetic X-intercept	0.0	0.0	0.0
Elbow Point	0.573317656186574	0.5444309135737296	0.5508937373398042
Synthetic Elbow Point	0.5066950327307322	0.5041903338140633	0.5064545074302387
Synthetic JS Distance	0.29386449490451394	0.2606668514302756	0.26577089471170084

Peak enrichment

Fraction of reads in peaks (FRiP)

FRiP for macs2 raw peaks

	rep1	rep2	rep3	rep1-pr1	rep2-pr1	rep3-pr1	rep1-pr2	rep2-pr2	rep3-pr2	pooled	pooled-pr1	pooled-pr2
Fraction of Reads in Peaks	0.1449935725350777	0.13571182598555592	0.13294893586076625	0.14945795907647372	0.14044001788276492	0.1408635130463024	0.148297321484799	0.13921656489969086	0.14321965997927685	0.13472665218227411	0.1322271025998867	0.13088013991648104

FRiP for overlap peaks

	rep1_vs_rep2	rep1_vs_rep3	rep2_vs_rep3	rep1-pr1_vs_rep1-pr2	rep2-pr1_vs_rep2-pr2	rep3-pr1_vs_rep3-pr2	pooled-pr1_vs_pooled-pr2
Fraction of Reads in Peaks	0.061338501176392626	0.06644962955513647	0.06154695272365803	0.08175726295671143	0.07484930755330965	0.06262105662626928	0.08964799207682374

FRiP for IDR peaks

	rep1_vs_rep2	rep1_vs_rep3	rep2_vs_rep3	rep1-pr1_vs_rep1-pr2	rep2-pr1_vs_rep2-pr2	rep3-pr1_vs_rep3-pr2	pooled-pr1_vs_pooled-pr2
Fraction of Reads in Peaks	0.006553651188159653	0.011176371082511051	0.01875814550463644	0.020457173198526957	0.022380593005642286	0.01814295110187451	0.030807184719715358

For macs2 raw peaks:

repX: Peak from true replicate X
repX-prY: Peak from Yth pseudoreplicates from replicate X
pooled: Peak from pooled true replicates (pool of rep1, rep2, ...)
pooled-pr1: Peak from 1st pooled pseudo replicate (pool of rep1-pr1, rep2-pr1, ...)
pooled-pr2: Peak from 2nd pooled pseudo replicate (pool of rep1-pr2, rep2-pr2, ...)

For overlap/IDR peaks:

repX_vs_repY: Comparing two peaks from true replicates X and Y
repX-pr1_vs_repX-pr2: Comparing two peaks from both pseudoreplicates from replicate X
pooled-pr1_vs_pooled-pr2: Comparing two peaks from 1st and 2nd pooled pseudo replicates